Fructokinase

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a fructokinase. The invention also relates to the construction of a chimeric gene encoding all or a portion of the fructokinase, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the fructokinase in a transformed host cell.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/244272, filed Oct. 30, 2000, the entire contents ofwhich are herein incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragmentsfructokinase in plants and seeds.

BACKGROUND OF THE INVENTION

[0003] Plastidic starch synthesis in plant storage tissues proceeds fromsucrose delivered via the phloem from the leaves. In one pathway,sucrose is converted into glucose and fructose by the action ofinvertase. Fructose is then phosphorylated to yield fructose6-phosphate, which is then converted to glucose 6-phosphate andeventually into starch by a number of enzymatic reactions.Phosphorylation of fructose may be carried out by fructokinase (EC2.7.1.4) or hexokinase (EC 2.7.1.1). However fructokinase whichspecifically phosphorylates fructose, may be more physiologicallyrelevant in phosphorylation of fructose than hexokinase which canphosphorylate a number of hexoses, since fructokinase has a much higheraffinity for fructose than hexokinase (Renz and Stitt (1993) Planta190:166-175).

[0004] Purification of fructokinase from a number of plant species,including potato (Renz and Stitt (1993) Planta 190:166-175), barley(Baysdorfer et al. (1989) J Plant Physiol 134:156-161), and corn(Doehlert (1989) Plant Physiol 89:1042-1048), indicated the existence ofmultiple isoforms which differed from each other in degree of inhibitionby fructose and/or specificity for nucleotide triphosphates.

[0005] cDNAs encoding two divergent fructokinases in tomato, Frk1 andFrk2, have been isolated and shown to be differentially regulated(Kanayama et al. (1997) Plant Physiol 113:1379-1384; Kanayama et al.(1998) Plant Physiol 117:85-90). Based on expression analysis, Frk1 isbelieved to play a housekeeping role, supplying glycolysis with fructose6-phosphate, whereas Frk2 may play a particularly important role instarch synthesis in tomato fruit (Kanayama et al. (1998) Plant Physiol117:85-90). Fructokinase has been similarly hypothesized to be importantin starch synthesis in other sink tissues, like the potato tuber (Daviesand Oparka (1985) J Plant Physiol 119:311-316; Ross et al. (1994)Physiol Plant 90:748-756).

[0006] Because of the importance of fructokinase in starch synthesis,there is accordingly a significant deal of interest in studyingfructokinase. Disclosed herein are nucleic acid fragments encodingfructokinase or portions thereof which may be of use in manipulatingfructokinase expression levels and/or enzyme activity in vivo, which maylead to altered levels of starch and/or lipid in the plant. Increasingfructokinase activity leads to more fructose 6-phosphate available forstarch synthesis.

SUMMARY OF THE INVENTION

[0007] The present invention relates to an isolated polynucleotidecomprising: (a) a first nucleotide sequence encoding a first polypeptidecomprising at least 100 amino acids, wherein the amino acid sequence ofthe first polypeptide and the amino acid sequence of SEQ ID NO: 4 haveat least 95% identity based on the Clustal alignment method, (b) asecond nucleotide sequence encoding a second polypeptide comprising atleast 200 amino acids, wherein the amino acid sequence of the secondpolypeptide and the amino acid sequence of SEQ ID NO: 10 have at least90% or 95% identity based on the Clustal alignment method, (c) a thirdnucleotide sequence encoding a third polypeptide comprising at least 250amino acids, wherein the amino acid sequence of the third polypeptideand the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO:12 have at least 80%, 85%, 90%, or 95% identity based on the Clustalalignment method, (d) a fourth nucleotide sequence encoding a fourthpolypeptide comprising at least 250 amino acids, wherein the amino acidsequence of the fourth polypeptide and the amino acid sequence of SEQ IDNO: 8 have at least 90% or 95% identity based on the Clustal alignmentmethod, or (e) the complement of the first, second, third, or fourthnucleotide sequence, wherein the complement and the first, second,third, or fourth nucleotide sequence contain the same number ofnucleotides and are 100% complementary. The first polypeptide preferablycomprises the amino acid sequence of SEQ ID NO: 4, the secondpolypeptide preferably comprises the amino acid sequence of SEQ ID NO:10, the third polypeptide preferably comprises the amino acid sequenceof SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 12, and the fourthpolypeptide preferably comprises the amino acid sequence of SEQ ID NO:8. The first nucleotide sequence preferably comprises the nucleotidesequence of SEQ ID NO: 3, the second nucleotide sequence preferablycomprises the nucleotide sequence of SEQ ID NO: 9, the third nucleotidesequence preferably comprises the nucleotide sequence of SEQ ID NO: 1,SEQ ID NO: 5, or SEQ ID NO: 11, and the fourth nucleotide sequencepreferably comprises the nucleotide sequence of SEQ ID NO: 7. The first,second, third, and fourth polypeptides preferably are fructokinases.

[0008] In a second embodiment, this invention relates to a vectorcomprising the polynucleotide of the present invention or a recombinantDNA construct comprising the polynucleotide of the present inventionoperably linked to at least one regulatory sequence.

[0009] In a third embodiment, the invention concerns a cell comprisingthe recombinant DNA construct of the present invention. The cell may bea eukaryotic cell such as a plant cell, or a prokaryotic cell such as abacterial cell.

[0010] In a fourth embodiment, the invention relates to a method oftransforming a cell by introducing into the cell a nucleic acidcomprising a polynucleotide of the present invention. The invention alsoconcerns a method for producing a plant comprising transforming a plantcell with a nucleic acid molecule comprising a polynucleotide of thepresent invention and regenerating a plant from the transformed plantcell. In a further embodiment, the seed from the transformed plant isincluded.

[0011] In a fifth embodiment, the present invention relates to anisolated polynucleotide fragment comprising a nucleotide sequencecomprised by any of the polynucleotides of the present invention,wherein the nucleotide sequence contains at least 30, 40, or 60nucleotides.

[0012] In a sixth embodiment the invention concerns a method forisolating a polypeptide encoded by the polynucleotide of the presentinvention comprising isolating the polypeptide from a cell containing arecombinant DNA construct comprising the polynucleotide operably linkedto a regulatory sequence.

[0013] In a seventh embodiment, the present invention relates to anisolated polypeptide comprising: (a) a first amino acid sequencecomprising at least 100 amino acids, wherein the first amino acidsequence and the amino acid sequence of SEQ ID NO: 4 have at least 95%identity based on the Clustal alignment method, (b) a second amino acidsequence comprising at least 200 amino acids, wherein the second aminoacid sequence and the amino acid sequence of SEQ ID NO: 10 have at least90% or 95% identity based on the Clustal alignment method, (c) a thirdamino acid sequence comprising at least 250 amino acids, wherein thethird amino acid sequence and the amino acid sequence of SEQ ID NO: 2,SEQ ID NO: 6, or SEQ ID NO: 12 have at least 80%, 85%, 90%, or 95%identity based on the Clustal alignment method, or (d) a fourth aminoacid sequence comprising at least 250 amino acids, wherein the fourthamino acid sequence and the amino acid sequence of SEQ ID NO: 8 have atleast 90% or 95% identity based on the Clustal alignment method. Thefirst amino acid sequence preferably comprises the amino acid sequenceof SEQ ID NO: 4, the second amino acid sequence preferably comprises theamino acid sequence of SEQ ID NO: 10, the third amino acid sequencepreferably comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:6, or SEQ ID NO: 12, and the fourth amino acid sequence preferablycomprises the amino acid sequence of SEQ ID NO: 8. The polypeptidepreferably is a fructokinase.

[0014] In an eighth embodiment, the present invention relates to avirus, preferably a baculovirus, comprising any of the isolatedpolynucleotides of the present invention or any of the recombinant DNAconstructs of the present invention.

[0015] In a ninth embodiment, the present invention relates to a methodof selecting an isolated polynucleotide that affects the level ofexpression of a fructokinase polypeptide or enzyme activity in a hostcell, preferably a plant cell, the method comprising the steps of: (a)constructing an isolated polynucleotide of the present invention or anisolated recombinant DNA construct of the present invention; (b)introducing the isolated polynucleotide or the isolated recombinant DNAconstruct into a host cell; (c) measuring the level of the fructokinasepolypeptide or enzyme activity in the host cell containing the isolatedpolynucleotide; and (d) comparing the level of the fructokinasepolypeptide or enzyme activity in the host cell containing the isolatedpolynucleotide with the level of the fructokinase polypeptide or enzymeactivity in the host cell that does not contain the isolatedpolynucleotide.

[0016] In a tenth embodiment, the present invention relates to a methodof obtaining a nucleic acid fragment encoding a substantial portion of afructokinase polypeptide, preferably a plant fructokinase polypeptide,comprising the steps of: synthesizing an oligonucleotide primercomprising a nucleotide sequence of at least one of 30 (preferably atleast one of 40, most preferably at least one of 60) contiguousnucleotides derived from a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 1, 3, 5, 7, 9, and 11, and the complement ofsuch nucleotide sequences; and amplifying a nucleic acid fragment(preferably a cDNA inserted in a cloning vector) using theoligonucleotide primer. The amplified nucleic acid fragment preferablywill encode a substantial portion of a fructokinase amino acid sequence.

[0017] In an eleventh embodiment, the present invention relates to amethod of obtaining a nucleic acid fragment encoding all or asubstantial portion of the amino acid sequence encoding a fructokinasepolypeptide comprising the steps of: probing a cDNA or genomic librarywith an isolated polynucleotide of the present invention; identifying aDNA clone that hybridizes with an isolated polynucleotide of the presentinvention; isolating the identified DNA clone; and sequencing the cDNAor genomic fragment that comprises the isolated DNA clone.

[0018] In a twelfth embodiment, the present invention concerns a methodfor positive selection of a transformed cell comprising: (a)transforming a host cell with the recombinant DNA construct of thepresent invention or an expression cassette of the present invention;and (b) growing the transformed host cell, preferably a plant cell, suchas a monocot or a dicot, under conditions which allow expression of thefructokinase polynucleotide in an amount sufficient to complement a nullmutant to provide a positive selection means.

[0019] In a thirteenth embodiment, the present invention relates to amethod of altering the level of expression of a fructokinase in a hostcell comprising: (a) transforming a host cell with a recombinant DNAconstruct of the present invention; and (b) growing the transformed hostcell under conditions that are suitable for expression of therecombinant DNA construct wherein expression of the recombinant DNAconstruct results in production of altered levels of the fructokinase inthe transformed host cell.

BRIEF DESCRIPTION OF THE DRAWING AND SEQUENCE LISTING

[0020] The invention can be more fully understood from the followingdetailed description and the accompanying drawing and Sequence Listingwhich form a part of this application.

[0021]FIG. 1 depicts an alignment of amino acid sequences offructokinase encoded by nucleotide sequences derived from corn clonecbn2n.pk0001.e11 (SEQ ID NO: 2), rice clone rl0n.pk087.f22 (SEQ ID NO:6), soybean clone sfl1.pk0123.d6 (SEQ ID NO: 8), wheat clonewyr1c.pk002.o12 (SEQ ID NO: 12), and Lycopersicon esculentum (NCBI GINo. 1915974; SEQ ID NO: 13). Amino acids which are conserved among alland at least two sequences with an amino acid at that position areindicated with an asterisk (*). Dashes are used by the program tomaximize alignment of the sequences.

[0022] Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825. TABLE 1 Fructokinase SEQ ID NO: (AminoProtein (Plant Source) Clone Designation (Nucleotide) Acid) Fructokinase(Corn) cbn2n.pk0001.e11 1 2 Fructokinase (Corn) p0010.cbpaf64r 3 4Fructokinase (Rice) rl0n.pk087.f22 5 6 Fructokinase (Soybean)sfl1.pk0123.d6 7 8 Fructokinase (Soybean) srr2c.pk002.i21 9 10Fructokinase (Wheat) wyr1c.pk002.o12 11 12

[0023] The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

[0024] In the context of this disclosure, a number of terms shall beutilized. The terms “polynucleotide”, “polynucleotide sequence”,“nucleic acid sequence”, and “nucleic acid fragment”/“isolated nucleicacid fragment” are used interchangeably herein. These terms encompassnucleotide sequences and the like. A polynucleotide may be a polymer ofRNA or DNA that is single- or double-stranded, that optionally containssynthetic, non-natural or altered nucleotide bases. A polynucleotide inthe form of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include at least 30contiguous nucleotides, preferably at least 40 contiguous nucleotides,most preferably at least 60 contiguous nucleotides derived from SEQ IDNOs: 1, 3, 5, 7, 9 or 11 or the complement of such sequences.

[0025] The term “isolated” refers to materials, such as nucleic acidmolecules and/or proteins, which are substantially free or otherwiseremoved from components that normally accompany or interact with thematerials in a naturally occurring environment. Isolated polynucleotidesmay be purified from a host cell in which they naturally occur.Conventional nucleic acid purification methods known to skilled artisansmay be used to obtain isolated polynucleotides. The term also embracesrecombinant polynucleotides and chemically synthesized polynucleotides.

[0026] The term “recombinant” means, for example, that a nucleic acidsequence is made by an artificial combination of two otherwise separatedsegments of sequence, e.g., by chemical synthesis or by the manipulationof isolated nucleic acids by genetic engineering techniques.

[0027] As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

[0028] As used herein, “substantially similar” refers to nucleic acidfragments wherein changes in one or more nucleotide bases results insubstitution of one or more amino acids, but do not affect thefunctional properties of the polypeptide encoded by the nucleotidesequence. “Substantially similar” also refers to nucleic acid fragmentswherein changes in one or more nucleotide bases does not affect theability of the nucleic acid fragment to mediate alteration of geneexpression by gene silencing through for example antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotides that do notsubstantially affect the functional properties of the resultingtranscript vis-à-vis the ability to mediate gene silencing or alterationof the functional properties of the resulting protein molecule. It istherefore understood that the invention encompasses more than thespecific exemplary nucleotide or amino acid sequences and includesfunctional equivalents thereof. The terms “substantially similar” and“corresponding substantially” are used interchangeably herein.

[0029] Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least 30 contiguous nucleotides, preferably at least 40contiguous nucleotides, most preferably at least 60 contiguousnucleotides derived from the instant nucleic acid fragment can beconstructed and introduced into a plant or plant cell. The level of thepolypeptide encoded by the unmodified nucleic acid fragment present in aplant or plant cell exposed to the substantially similar nucleicfragment can then be compared to the level of the polypeptide in a plantor plant cell that is not exposed to the substantially similar nucleicacid fragment.

[0030] For example, it is well known in the art that antisensesuppression and co-suppression of gene expression may be accomplishedusing nucleic acid fragments representing less than the entire codingregion of a gene, and by using nucleic acid fragments that do not share100% sequence identity with the gene to be suppressed. Moreover,alterations in a nucleic acid fragment which result in the production ofa chemically equivalent amino acid at a given site, but do not effectthe functional properties of the encoded polypeptide, are well known inthe art. Thus, a codon for the amino acid alanine, a hydrophobic aminoacid, may be substituted by a codon encoding another less hydrophobicresidue, such as glycine, or a more hydrophobic residue, such as valine,leucine, or isoleucine. Similarly, changes which result in substitutionof one negatively charged residue for another, such as aspartic acid forglutamic acid, or one positively charged residue for another, such aslysine for arginine, can also be expected to produce a functionallyequivalent product. Nucleotide changes which result in alteration of theN-terminal and C-terminal portions of the polypeptide molecule wouldalso not be expected to alter the activity of the polypeptide. Each ofthe proposed modifications is well within the routine skill in the art,as is determination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least 30 (preferably at least 40, mostpreferably at least 60) contiguous nucleotides derived from a nucleotidesequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9or 11 and the complement of such nucleotide sequences may be used toaffect the expression and/or function of a fructokinase polypeptide in ahost cell. A method of using an isolated polynucleotide to affect thelevel of expression of a polypeptide in a host cell (eukaryotic, such asplant or yeast, prokaryotic such as bacterial) may comprise the stepsof: constructing an isolated polynucleotide of the present invention oran isolated chimeric gene of the present invention; introducing theisolated polynucleotide or the isolated chimeric gene into a host cell;measuring the level of a polypeptide or enzyme activity in the host cellcontaining the isolated polynucleotide; and comparing the level of apolypeptide or enzyme activity in the host cell containing the isolatedpolynucleotide with the level of a polypeptide or enzyme activity in ahost cell that does not contain the isolated polynucleotide.

[0031] Moreover, substantially similar nucleic acid fragments may alsobe characterized by their ability to hybridize. Estimates of suchhomology are provided by either DNA-DNA or DNA-RNA hybridization underconditions of stringency as is well understood by those skilled in theart (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRLPress, Oxford, U.K.). Stringency conditions can be adjusted to screenfor moderately similar fragments, such as homologous sequences fromdistantly related organisms, to highly similar fragments, such as genesthat duplicate functional enzymes from closely related organisms.Post-hybridization washes determine stringency conditions. One set ofpreferred conditions uses a series of washes starting with 6× SSC, 0.5%SDS at room temperature for 15 min, then repeated with 2× SSC, 0.5% SDSat 45° C. for 30 min, and then repeated twice with 0.2× SSC, 0.5% SDS at50° C for 30 min. A more preferred set of stringent conditions useshigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2× SSG,0.5% SDS was increased to 60° C. Another preferred set of highlystringent conditions uses two final washes in 0.1× SSC, 0.1% SDS at 65°C.

[0032] Substantially similar nucleic acid fragments of the instantinvention may also be characterized by the percent identity of the aminoacid sequences that they encode to the amino acid sequences disclosedherein, as determined by algorithms commonly employed by those skilledin this art. Suitable nucleic acid fragments (isolated polynucleotidesof the present invention) encode polypeptides that are at least about70% identical, preferably at least about 80% identical to the amino acidsequences reported herein. Preferred nucleic acid fragments encode aminoacid sequences that are at least about 85% identical to the amino acidsequences reported herein. More preferred nucleic acid fragments encodeamino acid sequences that are at least about 90% identical to the aminoacid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are at least about 95%identical to the amino acid sequences reported herein. Suitable nucleicacid fragments not only have the above identities but typically encode apolypeptide having at least 50 amino acids, preferably at least 100amino acids, more preferably at least 150 amino acids, still morepreferably at least 200 amino acids, and most preferably at least 250amino acids. Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

[0033] A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also theexplanation of the BLAST algorithm on the world wide web site for theNational Center for Biotechnology Information at the National Library ofMedicine of the National Institutes of Health). In general, a sequenceof ten or more contiguous amino acids or thirty or more contiguousnucleotides is necessary in order to putatively identify a polypeptideor nucleic acid sequence as homologous to a known protein or gene.Moreover, with respect to nucleotide sequences, gene-specificoligonucleotide probes comprising 30 or more contiguous nucleotides maybe used in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12 or more nucleotides may be used as amplificationprimers in PCR in order to obtain a particular nucleic acid fragmentcomprising the primers. Accordingly, a “substantial portion” of anucleotide sequence comprises a nucleotide sequence that will affordspecific identification and/or isolation of a nucleic acid fragmentcomprising the sequence. The instant specification teaches amino acidand nucleotide sequences encoding polypeptides that comprise one or moreparticular plant proteins. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantialportion of the disclosed sequences for purposes known to those skilledin this art. Accordingly, the instant invention comprises the completesequences as reported in the accompanying Sequence Listing, as well assubstantial portions of those sequences as defined above.

[0034] “Codon degeneracy” refers to divergence in the genetic codepermitting variation of the nucleotide sequence without effecting theamino acid sequence of an encoded polypeptide. Accordingly, the instantinvention relates to any nucleic acid fragment comprising a nucleotidesequence that encodes all or a substantial portion of the amino acidsequences set forth herein. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing anucleic acid fragment for improved expression in a host cell, it isdesirable to design the nucleic acid fragment such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

[0035] “Synthetic nucleic acid fragments” can be assembled fromoligonucleotide building blocks that are chemically synthesized usingprocedures known to those skilled in the art. These building blocks areligated and annealed to form larger nucleic acid fragments which maythen be enzymatically assembled to construct the entire desired nucleicacid fragment. “Chemically synthesized”, as related to a nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of the nucleotide sequence to reflectthe codon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

[0036] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign-gene” refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

[0037] “Coding sequence” refers to a nucleotide sequence that codes fora specific amino acid sequence. “Regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

[0038] “Promoter” refers to a nucleotide sequence capable of controllingthe expression of a coding sequence or functional RNA. In general, acoding sequence is located 3′ to a promoter sequence. The promotersequence consists of proximal and more distal upstream elements, thelatter elements often referred to as enhancers. Accordingly, an“enhancer” is a nucleotide sequence which can stimulate promoteractivity and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter. Promoters may be derived in their entirety from a native gene,or may be composed of different elements derived from differentpromoters found in nature, or may even comprise synthetic nucleotidesegments. It is understood by those skilled in the art that differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental conditions. Promoters which cause a nucleic acidfragment to be expressed in most cell types at most times are commonlyreferred to as “constitutive promoters”. New promoters of various typesuseful in plant cells are constantly being discovered; numerous examplesmay be found in the compilation by Okamuro and Goldberg (1989)Biochemistry of Plants 15:1-82. It is further recognized that since inmost cases the exact boundaries of regulatory sequences have not beencompletely defined, nucleic acid fragments of different lengths may haveidentical promoter activity.

[0039] “Translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

[0040] “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

[0041] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to DNA that is complementary toand derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Klenowfragment of DNA polymerase I. “Sense-RNA” refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

[0042] The term “operably linked” refers to the association of two ormore nucleic acid fragments on a single polynucleotide so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

[0043] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid fragment of the invention. Expression mayalso refer to translation of mRNA into a polypeptide. “Antisenseinhibition” refers to the production of antisense RNA transcriptscapable of suppressing the expression of the target protein.“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Co-suppression” refers to the production ofsense RNA transcripts capable of suppressing the expression of identicalor substantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference).

[0044] A “protein” or “polypeptide” is a chain of amino acids arrangedin a specific order determined by the coding sequence in apolynucleotide encoding the polypeptide. Each protein or polypeptide hasa unique function.

[0045] “Altered levels” or “altered expression” refers to the productionof gene product(s) in transgenic organisms in amounts or proportionsthat differ from that of normal or non-transformed organisms.

[0046] “Mature protein” or the term “mature” when used in describing aprotein refers to a post-translationally processed polypeptide; i.e.,one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor protein” or the term“precursor” when used in describing a protein refers to the primaryproduct of translation of mRNA; i.e., with pre- and propeptides stillpresent. Pre- and propeptides may be but are not limited tointracellular localization signals.

[0047] A “chloroplast transit peptide” is an amino acid sequence whichis translated in conjunction with a protein and directs the protein tothe chloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

[0048] “Transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” organisms. Examples ofmethods of plant transformation include Agrobacterium-mediatedtransformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Thus, isolated polynucleotides of thepresent invention can be incorporated into recombinant constructs,typically DNA constructs, capable of introduction into and replicationin a host cell. Such a construct can be a vector that includes areplication system and sequences that are capable of transcription andtranslation of a polypeptide-encoding sequence in a given host cell. Anumber of vectors suitable for stable transfection of plant cells or forthe establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Flevin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

[0049] Standard recombinant DNA and molecular cloning techniques usedherein are well known in the art and are described more fully inSambrook et al. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter“Maniatis”).

[0050] “PCR” or “polymerase chain reaction” is well known by thoseskilled in the art as a technique used for the amplification of specificDNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

[0051] The present invention concerns an isolated polynucleotidecomprising a nucleotide sequence encoding a fructokinase polypeptideselected from the group consisting of: (a) a first nucleotide sequenceencoding a polypeptide of at least 100 amino acids having at least 95%identity based on the Clustal method of alignment when compared to apolypeptide of SEQ ID NO: 4, (b) a second nucleotide sequence encoding apolypeptide of at least 200 amino acids having at least 90% identitybased on the Clustal method of alignment when compared to a polypeptideof SEQ ID NO: 10, (c) a third nucleotide sequence encoding a polypeptideof at least 250 amino acids having at least 80% identity based on theClustal method of alignment when compared to a polypeptide selected fromthe group consisting of SEQ ID NOs: 2, 6, and 12, (d) a fourthnucleotide sequence encoding a polypeptide of at least 250 amino acidshaving at least 90% identity based on the Clustal method of alignmentwhen compared to a polypeptide of SEQ ID NO: 8, and (e) a fifthnucleotide sequence comprising the complement of the first, second,third, or fourth nucleotide sequence.

[0052] Preferably, the first nucleotide sequence comprises SEQ ID NO: 3,the second nucleotide sequence comprises SEQ ID NO: 9, the thirdnucleotide sequence comprises SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO:11, and the fourth nucleotide sequence comprises SEQ ID NO: 7.

[0053] This invention also relates to the isolated complement of suchpolynucleotides, wherein the complement and the polynucleotide consistof the same number of nucleotides, and the nucleotide sequences of thecomplement and the polynucleotide have 100% complementarity.

[0054] Nucleic acid fragments encoding at least a portion of severalfructokinases have been isolated and identified by comparison of randomplant cDNA sequences to public databases containing nucleotide andprotein sequences using the BLAST algorithms well known to those skilledin the art. The nucleic acid fragments of the instant invention may beused to isolate cDNAs and genes encoding homologous proteins from thesame or other plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

[0055] For example, genes encoding other fructokinases, either as cDNAsor genomic DNAs, could be isolated directly by using all or a portion ofthe instant nucleic acid fragments as DNA hybridization probes to screenlibraries from any desired plant employing methodology well known tothose skilled in the art. Specific oligonucleotide probes based upon theinstant nucleic acid sequences can be designed and synthesized bymethods known in the art (Maniatis). Moreover, an entire sequence can beused directly to synthesize DNA probes by methods known to the skilledartisan such as random primer DNA labeling, nick translation,end-labeling techniques, or RNA probes using available in vitrotranscription systems. In addition, specific primers can be designed andused to amplify a part or all of the instant sequences. The resultingamplification products can be labeled directly during amplificationreactions or labeled after amplification reactions, and used as probesto isolate full length cDNA or genomic fragments under conditions ofappropriate stringency.

[0056] In addition, two short segments of the instant nucleic acidfragments may be used in polymerase chain reaction protocols to amplifylonger nucleic acid fragments encoding homologous genes from DNA or RNA.The polymerase chain reaction may also be performed on a library ofcloned nucleic acid fragments wherein the sequence of one primer isderived from the instant nucleic acid fragments, and the sequence of theother primer takes advantage of the presence of the polyadenylic acidtracts to the 3′ end of the mRNA precursor encoding plant genes.Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci.USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies ofthe region between a single point in the transcript and the 3′ or 5′end. Primers oriented in the 3′ and 5′ directions can be designed fromthe instant sequences. Using commercially available 3′ RACE or 5′ RACEsystems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Oharaet al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989)Science 243:217-220). Products generated by the 3′ and 5′ RACEprocedures can be combined to generate full-length cDNAs (Frohman andMartin (1989) Techniques 1:165). Consequently, a polynucleotidecomprising a nucleotide sequence of at least 30 (preferably at least 40,most preferably at least 60) contiguous nucleotides derived from anucleotide sequence selected from the group consisting of SEQ ID NOs: 1,3, 5, 7, 9, and 11 and the complement of such nucleotide sequences maybe used in such methods to obtain a nucleic acid fragment encoding asubstantial portion of an amino acid sequence of a polypeptide.

[0057] Availability of the instant nucleotide and deduced amino acidsequences facilitates immunological screening of cDNA expressionlibraries. Synthetic peptides representing portions of the instant aminoacid sequences may be synthesized. These peptides can be used toimmunize animals to produce polyclonal or monoclonal antibodies withspecificity for peptides or proteins comprising the amino acidsequences. These antibodies can be then be used to screen cDNAexpression libraries to isolate full-length cDNA clones of interest(Lerner (1984) Adv. Immunol 36:1-34; Maniatis).

[0058] In another embodiment, this invention concerns viruses and hostcells comprising either the chimeric genes of the invention as describedherein or an isolated polynucleotide of the invention as describedherein. Examples of host cells which can be used to practice theinvention include, but are not limited to, yeast, bacteria, and plants.

[0059] As was noted above, the nucleic acid fragments of the instantinvention may be used to create transgenic plants in which the disclosedpolypeptides are present at higher or lower levels than normal or incell types or developmental stages in which they are not normally found.This would have the effect of altering the level of fructose 6-phosphatein those cells, which may lead to altered levels of starch and/or lipidin the plant.

[0060] Two different fructokinase genes, Frk1 and Frk2, have beenisolated from tomato and the advantages associated with increasing ordecreasing the levels of fructokinase polypeptides have been described(U.S. Pat. No. 6031154). Starch biosynthesis in storage tissues such astubers, roots and seeds requires fructokinase activity to provide theappropriate substrates. In some tissues, fructokinase has been suggestedto be rate-governing for substrate delivery; consequently,overexpression of fructokinase would be useful for promoting starchbiosynthesis. Additionally, inhibition of fructokinase activity inparticular tissues, e.g., seeds, roots, tubers, leaves, flowers and thelike, would be useful in suppressing the conversion of fructose tofructose-6-phosphate. This would result in the accumulation of fructosein those tissues, which would be sweeter as a result.

[0061] An enzymatic assay can be used to determine the level offructokinase activity (U.S. Pat. No. 6031154; Huber and Kakzawa (1985)Plant Physiol 81:1008). One skilled in the art would recognize thatother assays could be used to determine the level of fructokinasepolypeptide. These assays include, but are not limited to, immunoassays,electrophoresis detection assays (either with staining or westernblotting), and carbohydrate detection assays.

[0062] Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.The chimeric gene may comprise promoter sequences and translation leadersequences derived from the same genes. 3′ Non-coding sequences encodingtranscription termination signals may also be provided. The instantchimeric gene may also comprise one or more introns in order tofacilitate gene expression.

[0063] Plasmid vectors comprising the instant isolated polynucleotide(or chimeric gene) may be constructed. The choice of plasmid vector isdependent upon the method that will be used to transform host plants.The skilled artisan is well aware of the genetic elements that must bepresent on the plasmid vector in order to successfully transform, selectand propagate host cells containing the chimeric gene. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

[0064] For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by directing the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) with or without removingtargeting sequences that are already present. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of use may be discovered in the future.

[0065] It may also be desirable to reduce or eliminate expression ofgenes encoding the instant polypeptides in plants for some applications.In order to accomplish this, a chimeric gene designed for co-suppressionof the instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

[0066] Molecular genetic solutions to the generation of plants withaltered gene expression have a decided advantage over more traditionalplant breeding approaches. Changes in plant phenotypes can be producedby specifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression of aspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

[0067] The person skilled in the art will know that specialconsiderations are associated with the use of antisense or cosuppressiontechnologies in order to reduce expression of particular genes. Forexample, the proper level of expression of sense or antisense genes mayrequire the use of different chimeric genes utilizing differentregulatory elements known to the skilled artisan. Once transgenic plantsare obtained by one of the methods described above, it will be necessaryto screen individual transgenics for those that most effectively displaythe desired phenotype. Accordingly, the skilled artisan will developmethods for screening large numbers of transformants. The nature ofthese screens will generally be chosen on practical grounds. Forexample, one can screen by looking for changes in gene expression byusing antibodies specific for the protein encoded by the gene beingsuppressed, or one could establish assays that specifically measureenzyme activity. A preferred method will be one which allows largenumbers of samples to be processed rapidly, since it will be expectedthat a large number of transformants will be negative for the desiredphenotype.

[0068] In another embodiment, the present invention concerns apolypeptide selected from the group consisting of: (a) a polypeptide ofat least 100 amino acids having at least 95% identity based on theClustal method of alignment when compared to a polypeptide of SEQ ID NO:4, (b) a polypeptide of at least 200 amino acids having at least 90%identity based on the Clustal method of alignment when compared to apolypeptide of SEQ ID NO: 10, (c) a polypeptide of at least 250 aminoacids having at least 80% identity based on the Clustal method ofalignment when compared to a polypeptide selected from the groupconsisting of SEQ ID NOs: 2,6, and 12, and (d) a polypeptide of at least250 amino acids having at least 90% identity based on the Clustal methodof alignment when compared to a polypeptide of SEQ ID NO: 8.

[0069] The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to these proteins by methods wellknown to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded fructokinase. An example of a vector for high levelexpression of the instant polypeptides in a bacterial host is provided(Example 6).

[0070] All or a substantial portion of the polynucleotides of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and used asmarkers for traits linked to those genes. Such information may be usefulin plant breeding in order to develop lines with desired phenotypes. Forexample, the instant nucleic acid fragments may be used as restrictionfragment length polymorphism (RFLP) markers. Southern blots (Maniatis)of restriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet32:314-331).

[0071] The production and use of plant gene-derived probes for use ingenetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol.Biol. Reporter 4:37-41. Numerous publications describe genetic mappingof specific cDNA clones using the methodology outlined above orvariations thereof. For example, F2 intercross populations, backcrosspopulations, randomly mated populations, near isogenic lines, and othersets of individuals may be used for mapping. Such methodologies are wellknown to those skilled in the art.

[0072] Nucleic acid probes derived from the instant nucleic acidsequences may also be used for physical mapping (i.e., placement ofsequences on physical maps; see Hoheisel et al. In: Nonmammalian GenomicAnalysis: A Practical Guide, Academic press 1996, pp. 319-346, andreferences cited therein).

[0073] Nucleic acid probes derived from the instant nucleic acidsequences may be used in direct fluorescence in situ hybridization(FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although currentmethods of FISH mapping favor use of large clones (several to severalhundred KB; see Laan et al. (1995) Genome Res. 5:13-20), improvements insensitivity may allow performance of FISH mapping using shorter probes.

[0074] A variety of nucleic acid amplification-based methods of geneticand physical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) NatGenet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

[0075] Loss of function mutant phenotypes may be identified for theinstant cDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptide.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptide can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptide disclosed herein.

EXAMPLES

[0076] The present invention is further defined in the followingExamples, in which parts and percentages are by weight and degrees areCelsius, unless otherwise stated. It should be understood that theseExamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions. Thus, variousmodifications of the invention in addition to those shown and describedherein will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

[0077] The disclosure of each reference set forth herein is incorporatedherein by reference in its entirety.

EXAMPLE 1 Composition of cDNA Libraries: Isolation and Sequencing ofcDNA Clones

[0078] cDNA libraries representing mRNAs from various corn (Zea mays),rice (Oryza saliva), soybean (Glycine max), and wheat (Triticumaestivum) tissues were prepared. The characteristics of the librariesare described below. TABLE 2 cDNA Libraries from Corn, Rice, Soybean,and Wheat Library Tissue Clone cbn2n Corn Developing Kernel Two DaysAfter cbn2n.pk0001.e11 Pollination* p0010 Corn Log Phase SuspensionCells Treated p0010.cbpaf64r With A23187 ® ** to Induce Mass Apoptosisrl0n Rice 15 Day Old Leaf* rl0n.pk087.f22 sfl1 Soybean Immature Flowersfl1.pk0123.d6 srr2c Soybean 8-Day-Old Root srr2c.pk002.i21 wyr1c WheatYellow Rust Infested Tissue wyr1c.pk002.o12

[0079] cDNA libraries may be prepared by any one of many methodsavailable. For example, the cDNAs may be introduced into plasmid vectorsby first preparing the cDNA libraries in Uni-ZAP™ XR vectors accordingto the manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif.). The Uni-ZAP™ XR libraries are converted into plasmid librariesaccording to the protocol provided by Stratagene. Upon conversion, cDNAinserts will be contained in the plasmid vector pBluescript. Inaddition, the cDNAs may be introduced directly into precut Bluescript IISK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs),followed by transfection into DH10B cells according to themanufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts arein plasmid vectors, plasmid DNAs are prepared from randomly pickedbacterial colonies containing recombinant pBluescript plasmids, or theinsert cDNA sequences are amplified via polymerase chain reaction usingprimers specific for vector sequences flanking the inserted cDNAsequences. Amplified insert DNAs or plasmid DNAs are sequenced indye-primer sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

[0080] Full-insert sequence (FIS) data is generated utilizing a modifiedtransposition protocol. Clones identified for FIS are recovered fromarchived glycerol stocks as single colonies, and plasmid DNAs areisolated via alkaline lysis. Isolated DNA templates are reacted withvector primed M13 forward and reverse oligonucleotides in a PCR-basedsequencing reaction and loaded onto automated sequencers. Confirmationof clone identification is performed by sequence alignment to theoriginal EST sequence from which the FIS request is made.

[0081] Confirmed templates are transposed via the Primer Islandtransposition kit (PE Applied Biosystems, Foster City, Calif.) which isbased upon the Saccharomyces cerevisiae Ty1 transposable element (Devineand Boeke (1994) Nucleic Acids Res. 22:3765-3772). The in vitrotransposition system places unique binding sites randomly throughout apopulation of large DNA molecules. The transposed DNA is then used totransform DH10B electro-competent cells (Gibco BRL/Life Technologies,Rockville, Md.) via electroporation. The transposable element containsan additional selectable marker (named DHFR; Fling and Richards (1983)Nucleic Acids Res. 11:5147-5158), allowing for dual selection on agarplates of only those subclones containing the integrated transposon.Multiple subclones are randomly selected from each transpositionreaction, plasmid DNAs are prepared via alkaline lysis, and templatesare sequenced (ABI Prism dye-terminator ReadyReaction mix) outward fromthe transposition event site, utilizing unique primers specific to thebinding sites within the transposon.

[0082] Sequence data is collected (ABI Prism Collections) and assembledusing Phred/Phrap (P. Green, University of Washington, Seattle).Phrep/Phrap is a public domain software program which re-reads the ABIsequence data, re-calls the bases, assigns quality values, and writesthe base calls and quality values into editable output files. The Phrapsequence assembly program uses these quality values to increase theaccuracy of the assembled sequence contigs. Assemblies are viewed by theConsed sequence editor (D. Gordon, University of Washington, Seattle).

[0083] In some of the clones the cDNA fragment corresponds to a portionof the 3′ -terminus of the gene and does not cover the entire openreading frame. In order to obtain the upstream information one of twodifferent protocols are used. The first of these methods results in theproduction of a fragment of DNA containing a portion of the desired genesequence while the second method results in the production of a fragmentcontaining the entire open reading frame. Both of these methods use tworounds of PCR amplification to obtain fragments from one or morelibraries. The libraries some times are chosen based on previousknowledge that the specific gene should be found in a certain tissue andsome times are randomly-chosen. Reactions to obtain the same gene may beperformed on several libraries in parallel or on a pool of libraries.Library pools are normally prepared using from 3 to 5 differentlibraries and normalized to a uniform dilution. In the first round ofamplification both methods use a vector-specific (forward) primercorresponding to a portion of the vector located at the 5′ -terminus ofthe clone coupled with a gene-specific (reverse) primer. The firstmethod uses a sequence that is complementary to a portion of the alreadyknown gene sequence while the second method uses a gene-specific primercomplementary to a portion of the 3′ -untranslated region (also referredto as UTR). In the second round of amplification a nested set of primersis used for both methods. The resulting DNA fragment is ligated into apBluescript vector using a commercial kit and following themanufacturer's protocol. This kit is selected from many available fromseveral vendors including Invitrogen (Carlsbad, Calif.), Promega Biotech(Madison, Wis.), and Gibco-BRL (Gaithersburg, Md.). The plasmid DNA isisolated by alkaline lysis method and submitted for sequencing andassembly using Phred/Phrap, as above.

EXAMPLE 2 Identification of cDNA Clones

[0084] cDNA clones encoding fructokinase were identified by conductingBLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol.Biol. 215:403-410; see also the explanation of the BLAST algorithm onthe world wide web site for the National Center for BiotechnologyInformation at the National Library of Medicine of the NationalInstitutes of Health) searches for similarity to sequences contained inthe BLAST “nr” database (comprising all non-redundant GenBank CDStranslations, sequences derived from the 3-dimensional structureBrookhaven Protein Data Bank, the last major release of the SWISS-PROTprotein sequence database, EMBL, and DDBJ databases). The cDNA sequencesobtained in Example 1 were analyzed for similarity to all publiclyavailable DNA sequences contained in the “nr” database using the BLASTNalgorithm provided by the National Center for Biotechnology Information(NCBI). The DNA sequences were translated in all reading frames andcompared for similarity to all publicly available protein sequencescontained in the “nr” database using the BLASTX algorithm (Gish andStates (1993) Nat Genet 3:266-272) provided by the NCBI. Forconvenience, the P-value (probability) of observing a match of a cDNAsequence to a sequence contained in the searched databases merely bychance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

[0085] ESTs submitted for analysis are compared to the genbank databaseas described above. ESTs that contain sequences more 5- or 3-prime canbe found by using the BLASTN algorithm (Altschul et al (1997) NucleicAcids Res. 25:3389-3402.) against the Du Pont proprietary databasecomparing nucleotide sequences that share common or overlapping regionsof sequence homology. Where common or overlapping sequences existbetween two or more nucleic acid fragments, the sequences can beassembled into a single contiguous nucleotide sequence, thus extendingthe original fragment in either the 5 or 3 prime direction. Once themost 5-prime EST is identified, its complete sequence can be determinedby Full Insert Sequencing as described in Example 1. Homologous genesbelonging to different species can be found by comparing the amino acidsequence of a known gene (from either a proprietary source or a publicdatabase) against an EST database using the TBLASTN algorithm. TheTBLASTN algorithm searches an amino acid query against a nucleotidedatabase that is translated in all 6 reading frames. This search allowsfor differences in nucleotide codon usage between different species, andfor codon degeneracy.

EXAMPLE 3 Characterization of cDNA Clones Encoding Fructokinase

[0086] The BLASTX search using the EST sequences from clones listed inTable 3 revealed similarity of the polypeptides encoded by the cDNAs tofructokinase from Arabidopsis thaliana (NCBI General Identifier (GI)Nos. 7434221 and 4589962) and Lycopersicon esculentum (NCBI GI No.1915974). Shown in Table 3 are the BLAST results for individual ESTs(“EST”), the sequences of the entire cDNA inserts comprising theindicated cDNA clones (“FIS”), the sequences of contigs assembled fromtwo or more ESTs (“Contig”), sequences of contigs assembled from an FISand one or more ESTs (“Contig*”), or sequences encoding an entireprotein derived from an FIS, a contig, or an FIS and PCR fragmentsequence (“CGS”): TABLE 3 BLAST Results for Sequences EncodingPolypeptides Homologous to Fructokinase BLAST Results Clone Status NCBIGI No. BLAST pLog Score cbn2n.pk0001.e11 (FIS) CGS 7434221 138.00p0010.cbpaf64r EST 4589962 40.22 rl0n.pk087.f22 (FIS) CGS 4589962 135.00sfl1.pk0123.d6 (FIS) CGS 1915974 157.00 srr2c.pk002.i21 FIS 1915974103.00 wyr1c.pk002.o12 (FIS) CGS 7434221 137.00

[0087]FIG. 1 presents an alignment of the amino acid sequences set forthin SEQ ID NOs: 2, 6, 8, and 12 and the Lycopersicon esculentum sequence(NCBI GI No. 1915974; SEQ ID NO: 13). The amino acid sequences of SEQ IDNOs: 2, 6, 8, and 12, shown in FIG. 1, are continuous open-readingframes predicted from the nucleotide sequences of SEQ ID NOs: 1, 5, 7,and 11, respectively. From the alignment of the amino acid sequences ofSEQ ID NOs: 2, 6, 8, and 12 with the Lycopersicon esculentum sequence(NCBI GI No. 1915974; SEQ ID NO: 13), it is apparent that the initialstart methionine codon for SEQ ID NOs: 2, 6, 8, and 12 are at amino acidpositions 37, 46, 24, and 1, respectively.

[0088] Pego and Smeekens (2000) Trends in Plant Science 5:531-536, havecompared the amino acid sequences of seven plant fructokinases fromArabidopsis, tomato, sugar beet and potato. They note four conserveddomains, present in the order of A1, B, A2 and A3. The amino acidsequences of SEQ ID NOs: 2, 6, 8 and 12, shown in FIG. 1, have theseconserved domains and they are present in the same order. Given in termsof the consensus amino acid positions of FIG. 1, the A1 domain is fromamino acid 93 to 113, the B domain is from amino acid 194 to 258, the A2domain is from amino acid 279 to 291, and the A3 domain is from aminoacid 312 to 325. The A1, A2 and A3 domains are signature domains for thepfkB family of carbohydrate kinases and the B domain is a regionspecific to fructokinases. The A1 motif is involved in ATP binding andthe B motif contains two sugar-binding domains.

[0089] The data in Table 4 represents a calculation of the percentidentity of the amino acid sequences set forth in SEQ ID NOs: 2, 6, 8,and 12 and the Lycopersicon esculentum sequence (NCBI GI No. 1915974;SEQ ID NO: 13). TABLE 4 Percent Identity of Amino Acid Sequences DeducedFrom the Nucleotide Sequences of cDNA Clones Encoding PolypeptidesHomologous to Fructokinase Percent Identity to SEQ ID NO. NCBI GI No.1915974; SEQ ID NO:13 2 66.2 6 69.2 8 83.8 12 67.1

[0090] Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores andprobabilities indicate that the nucleic acid fragments comprising theinstant cDNA clones encode a substantial portion of a fructokinase.These sequences represent the first monocot (corn, rice, and wheat)sequences indicated as encoding fructokinase known to Applicant. SoybeanESTs encoding fructokinase (e.g., NCBI GI No. 6747676) have beendisclosed previously.

EXAMPLE 4 Expression of Chimeric Genes in Monocot Cells

[0091] A chimeric gene comprising a cDNA encoding the instantpolypeptide in sense orientation with respect to the maize 27 kD zeinpromoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′end that is located 3′ to the cDNA fragment, can be constructed. ThecDNA fragment of this gene may be generated by polymerase chain reaction(PCR) of the cDNA clone using appropriate oligonucleotide primers.Cloning sites (Ncol or Smal) can be incorporated into theoligonucleotides to provide proper orientation of the DNA fragment wheninserted into the digested vector pML103 as described below.Amplification is then performed in a standard PCR. The amplified DNA isthen digested with restriction enzymes Ncol and Smal and fractionated onan agarose gel. The appropriate band can be isolated from the gel andcombined with a 4.9 kb Ncol-Smal fragment of the plasmid pML103. PlasmidpML103 has been deposited under the terms of the Budapest Treaty at ATCC(American Type Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209), and bears accession number ATCC 97366. The DNA segment frompML103 contains a 1.05 kb sally-Ncol promoter fragment of the maize 27kD zein gene and a 0.96 kb Smal-Sall fragment from the 3′ end of themaize 10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector andinsert DNA can be ligated at 150° C. overnight, essentially as described(Maniatis). The ligated DNA may then be used to transform E. coliXL1-Blue (Epicurian Coli XL-1 Blue™ ; Stratagene). Bacterialtransformants can be screened by restriction enzyme digestion of plasmidDNA and limited nucleotide sequence analysis using the dideoxy chaintermination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical).The resulting plasmid construct would comprise a chimeric gene encoding,in the 5′ to 3′ direction, the maize 27 kD zein promoter, a cDNAfragment encoding the instant polypeptide, and the 10 kD zein 3′ region.

[0092] The chimeric gene described above can then be introduced intocorn cells by the following procedure. Immature corn embryos can bedissected from developing caryopses derived from crosses of the inbredcorn lines H99 and LH132. The embryos are isolated 10 to 11 days afterpollination when they are 1.0 to 1.5 mm long. The embryos are thenplaced with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0093] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0094] The particle bombardment method (Klein et al. (1987) Nature327:70-73) may be used to transfer genes to the callus culture cells.According to this method, gold particles (1 μm in diameter) are coatedwith DNA using the following technique. Ten μg of plasmid DNAs are addedto 50 μL of a suspension of gold particles (60 mg per mL). Calciumchloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL ofa 1.0 M solution) are added to the particles. The suspension is vortexedduring the addition of these solutions. After 10 minutes, the tubes arebriefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed.The particles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

[0095] For bombardment, the embryogenic tissue is placed on filter paperover agarose-solidified N6 medium. The tissue is arranged as a thin lawnand covered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

[0096] Seven days after bombardment the tissue can be transferred to N6medium that contains bialophos (5 mg per liter) and lacks casein orproline. The tissue continues to grow slowly on this medium. After anadditional 2 weeks the tissue can be transferred to fresh N6 mediumcontaining bialophos. After 6 weeks, areas of about 1 cm in diameter ofactively growing callus can be identified on some of the platescontaining the bialophos-supplemented medium. These calli may continueto grow when sub-cultured on the selective medium.

[0097] Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

EXAMPLE 5 Expression of Chimeric Genes in Dicot Cells

[0098] A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptide in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′ ) from thetranslation initiation codon and about 1650 nucleotides downstream (3′ )from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Ncol (whichincludes the ATG translation initiation codon), Smal, Kpnl and Xbal. Theentire cassette is flanked by HindIII sites.

[0099] The cDNA fragment of this gene may be generated by polymerasechain reaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

[0100] Soybean embryos may then be transformed with the expressionvector comprising sequences encoding the instant polypeptide. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

[0101] Soybean embryogenic suspension cultures can be maintained in 35mL liquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

[0102] Soybean embryogenic suspension cultures may then be transformedby the method of particle gun bombardment (Klein et al. (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

[0103] A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptide and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

[0104] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

[0105] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. For each transformation experiment,approximately 5-10 plates of tissue are normally bombarded. Membranerupture pressure is set at 1100 psi and the chamber is evacuated to avacuum of 28 inches mercury. The tissue is placed approximately 3.5inches away from the retaining screen and bombarded three times.Following bombardment, the tissue can be divided in half and placed backinto liquid and cultured as described above.

[0106] Five to seven days post bombardment, the liquid media may beexchanged with fresh media, and eleven to twelve days post bombardmentwith fresh media containing 50 mg/mL hygromycin. This selective mediacan be refreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

EXAMPLE 6 Expression of Chimeric Genes in Microbial Cells

[0107] The cDNA encoding the instant polypeptide can be inserted intothe T7 E. coli expression vector pBT430. This vector is a derivative ofpET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoRI and HindIII sites in pET-3a attheir original positions. An oligonucleotide adaptor containing EcoRIand Hind III sites was inserted at the BamHI site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Ndel site at the position oftranslation initiation was converted to an Ncol site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0108] Plasmid DNA containing a cDNA may be appropriately digested torelease a nucleic acid fragment encoding the protein. This fragment maythen be purified on a 1% low melting agarose gel. Buffer and agarosecontain 10 μg/ml ethidium bromide for visualization of the DNA fragment.The fragment can then be purified from the agarose gel by digestion withGELase™ (Epicentre Technologies, Madison, Wis.) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptide are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

[0109] For high level expression, a plasmid clone with the cDNA insertin the correct orientation relative to the T7 promoter can betransformed into E. coli strain BL21 (DE3) (Studier et al. (1986) J.Mol. Biol. 189:113-130). Cultures are grown in LB medium containingampicillin (100 mg/L) at 25° C. At an optical density at 600 nm ofapproximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can beadded to a final concentration of 0.4 mM and incubation can be continuedfor 3 h at 250. Cells are then harvested by centrifugation andre-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTTand 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glassbeads can be added and the mixture sonicated 3 times for about 5 secondseach time with a microprobe sonicator. The mixture is centrifuged andthe protein concentration of the supernatant determined. One μg ofprotein from the soluble fraction of the culture can be separated bySDS-polyacrylamide gel electrophoresis. Gels can be observed for proteinbands migrating at the expected molecular weight.

1 13 1 1493 DNA Zea mays 1 gcacgagaca atcgcctcgc cttcccttcc ccaccagcccgtctctctct tctctctgtc 60 tctcttctcg taaccgcgtc cacctcgcag cagcaagcaagcgcgaccaa atggcgcctc 120 taggagacgg cggagctgct gccgcggcgg cgtccaacaacctggtggtg tcgttcggcg 180 agatgctgat cgacttcgtc cccgacgtgg ccgggctgtcgctggccgag tcgggcggct 240 tcgtcaaggc ccccggcggc gcgcccgcca acgtcgcctgcgccatcgcc aagctcggcg 300 gatcctccgc cttcgtcggc aagttcggcg acgacgagttcgggcacatg ttggtgaaca 360 tcctgaagca gaacaacgtg aactcggagg ggtgcctgttcgacaagcac gcgcggacgg 420 cgctggcctt cgtgacgctc aagcacgacg gggagcgcgagttcatgttc tacaggaacc 480 cgagcgcgga catgctgctg acggaggcgg agctggacctgggcctggtg cggcgcgcca 540 aggtgttcca ctacggctcc atctcgctca tctccgagccgtgccgctcg gcgcacatgg 600 ccgccatgcg cgcagccaag gccgcgggcg tgctctgctcctacgacccc aacgtgcgcc 660 tcccgctctg gccgtcgccc gacgccgcac gcgagggcatcctcagcatc tggaaggagg 720 ccgacttcat caaggtcagc gacgacgagg tggccttcctcacgcgcggg gacgccaacg 780 acgagaagaa cgtgctgtcc ctgtggtttg acgggctcaagctgctcgtc gtcaccgacg 840 gggacaaggg atgcaggtac ttcaccaagg acttcaagggcagcgtgccc ggcttcaagg 900 tcgacaccgt cgacaccacc ggcgccggcg acgccttcgtcggctccctc ctcgtcaacg 960 tcgccaagga cgactccatc ttccacaacg aggagaagctccgcgaggct ctcaagttct 1020 ccaacgcctg cggcgccatc tgcaccacca agaagggcgccatcccggcg ctgcccacgg 1080 tcgccaccgc ccaggacctc atcgccaagg ccaactagatggccgcacgc cccgccgttc 1140 caccacgtca ctgtcccccg ccgccccgcc cctcgtcgtcgacgtcctcg gtttcggttc 1200 attaggtaga tcgagtctta gcgtccgtct ctgcgcctctacgctgagac ggtttgtttg 1260 ggttaattaa gttagctttc gtggagattt cgccccggggcatcaataaa atgttggcat 1320 gcgtggtggg atgctatcct ttttttttat tttattttattttattttta gcttggatca 1380 gttggggttt tgaacattgc tagtgtcgtg tgattgggaaggctaatgtg atgccttcga 1440 tgcagagttt tcaatgaatg ccttggtgca aacgtaaaaaaaaaaaaaaa aaa 1493 2 371 PRT Zea mays 2 Thr Arg Gln Ser Pro Arg Leu ProPhe Pro Thr Ser Pro Ser Leu Ser 1 5 10 15 Ser Leu Cys Leu Ser Ser ArgAsn Arg Val His Leu Ala Ala Ala Ser 20 25 30 Lys Arg Asp Gln Met Ala ProLeu Gly Asp Gly Gly Ala Ala Ala Ala 35 40 45 Ala Ala Ser Asn Asn Leu ValVal Ser Phe Gly Glu Met Leu Ile Asp 50 55 60 Phe Val Pro Asp Val Ala GlyLeu Ser Leu Ala Glu Ser Gly Gly Phe 65 70 75 80 Val Lys Ala Pro Gly GlyAla Pro Ala Asn Val Ala Cys Ala Ile Ala 85 90 95 Lys Leu Gly Gly Ser SerAla Phe Val Gly Lys Phe Gly Asp Asp Glu 100 105 110 Phe Gly His Met LeuVal Asn Ile Leu Lys Gln Asn Asn Val Asn Ser 115 120 125 Glu Gly Cys LeuPhe Asp Lys His Ala Arg Thr Ala Leu Ala Phe Val 130 135 140 Thr Leu LysHis Asp Gly Glu Arg Glu Phe Met Phe Tyr Arg Asn Pro 145 150 155 160 SerAla Asp Met Leu Leu Thr Glu Ala Glu Leu Asp Leu Gly Leu Val 165 170 175Arg Arg Ala Lys Val Phe His Tyr Gly Ser Ile Ser Leu Ile Ser Glu 180 185190 Pro Cys Arg Ser Ala His Met Ala Ala Met Arg Ala Ala Lys Ala Ala 195200 205 Gly Val Leu Cys Ser Tyr Asp Pro Asn Val Arg Leu Pro Leu Trp Pro210 215 220 Ser Pro Asp Ala Ala Arg Glu Gly Ile Leu Ser Ile Trp Lys GluAla 225 230 235 240 Asp Phe Ile Lys Val Ser Asp Asp Glu Val Ala Phe LeuThr Arg Gly 245 250 255 Asp Ala Asn Asp Glu Lys Asn Val Leu Ser Leu TrpPhe Asp Gly Leu 260 265 270 Lys Leu Leu Val Val Thr Asp Gly Asp Lys GlyCys Arg Tyr Phe Thr 275 280 285 Lys Asp Phe Lys Gly Ser Val Pro Gly PheLys Val Asp Thr Val Asp 290 295 300 Thr Thr Gly Ala Gly Asp Ala Phe ValGly Ser Leu Leu Val Asn Val 305 310 315 320 Ala Lys Asp Asp Ser Ile PheHis Asn Glu Glu Lys Leu Arg Glu Ala 325 330 335 Leu Lys Phe Ser Asn AlaCys Gly Ala Ile Cys Thr Thr Lys Lys Gly 340 345 350 Ala Ile Pro Ala LeuPro Thr Val Ala Thr Ala Gln Asp Leu Ile Ala 355 360 365 Lys Ala Asn 3703 430 DNA Zea mays unsure (293) n = A, C, G or T 3 gcgacgacga gttcggccgcatgctcgccg ccatcctccg cgacaacggc gtcgacggcg 60 gcggcgtcgt cttcgacgcgggcgcgcgca ccgccttgcc ttcgtcaccc tgcgcgccga 120 cggcgagcgc gagttcatgttctaccgcaa ccccagcgcc gacatgctcc tcactgccga 180 cgagctcaac gtcgggctcatccggagggc tgcggtcttt cactacggat caataagctt 240 gattgctgag ccttgccggacagcacatct ccgtgccatg gaaattgcca aanaggctgg 300 tgcactgctc tcttacgacccaaacctgag ggaggcactt tggccatccc gtgaggaggc 360 ccgcacccag atcttgagcattgggaccag gcagatatcg tcaaggtcag cgaagtcgag 420 cttgagtttt 430 4 101 PRTZea mays UNSURE (72) Xaa = ANY AMINO ACID 4 Gly Arg Ala His Arg Leu AlaPhe Val Thr Leu Arg Ala Asp Gly Glu 1 5 10 15 Arg Glu Phe Met Phe TyrArg Asn Pro Ser Ala Asp Met Leu Leu Thr 20 25 30 Ala Asp Glu Leu Asn ValGly Leu Ile Arg Arg Ala Ala Val Phe His 35 40 45 Tyr Gly Ser Ile Ser LeuIle Ala Glu Pro Cys Arg Thr Ala His Leu 50 55 60 Arg Ala Met Glu Ile AlaLys Xaa Ala Gly Ala Leu Leu Ser Tyr Asp 65 70 75 80 Pro Asn Leu Arg GluAla Leu Trp Pro Ser Arg Glu Glu Ala Arg Thr 85 90 95 Gln Ile Leu Ser Ile100 5 1553 DNA Oryza sativa 5 gcacgagctt acactcatct catctcatctcaccctcgcc gcgcgccgag gaagacgcgc 60 atctcctctc tccctctata taagcgcgcgcctcgccacc tcacccgaag aaattcccca 120 ccattccatc tctctctctc tcgaatcttgatctctctct ttcatcgcct cttgtgttcg 180 cgcgcgcgag cagggtggtt gttgttggtgggggtgcaat ggcggggagg agcgagctgg 240 tggtgagctt cggggagatg ctgatagacttcgtgccgac ggtggcgggg gtgtcgctgg 300 cggaggcgcc ggcgttcgtc aaggcgccagggggggcgcc ggcgaacgtg gccatcgcgg 360 tggcgcggct cggcggcggg gccgcgttcgtcggcaagct gggggacgac gagttcgggc 420 ggatgctcgc ggccatcctc cgcgacaacggcgtcgacga cggcggggtc gtgttcgacg 480 ccggggcgcg caccgcgctc gccttcgtcaccctccgcgc cgacggggag cgcgagttca 540 tgttctaccg caaccccagc gccgacatgctcctcaccca cgccgagctc aacgtcgagc 600 tcatcaagag ggctgccgtc ttccattatggatcaataag cttgatagct gagccctgcc 660 ggtcagcaca tttgcgtgcc atggagattgcgaaagaagc tggtgcgctg ctatcttatg 720 acccgaatct cagggaggca ttgtggccctcccgtgagga ggctcgcacc aagatcttga 780 gcatctggga ccaggcagac attgtcaaggtcagcgaggt cgagcttgag ttcttgaccg 840 gcattgactc agtagaggat gatgttgtcatgaagctatg gcgccctacc atgaagctcc 900 tccttgtgac tcttggagat caaggatgcaagtactatgc cagggatttc cgcggagctg 960 tcccatccta caaagtacag caagttgatacaacaggtgc aggtgatgcg tttgttggtg 1020 ctctgctgcg aagaattgtc caggatccatcatcgttgca agatcagaag aagcttgagg 1080 aagcgattaa atttgccaat gcgtgcggagcaatcaccgc cacaaagaaa ggggcaatcc 1140 catcactgcc caccgaagtt gaggtcttgaagttgatgga gagtgcttag atcgatcagt 1200 agcattatgg tcactagctt cagcttccgcaaattgtatt gtatgctgat ctggatcagg 1260 agcagggggg tactccaaga tgcctgcctttttgttgcca acttcccttc ctggcaggat 1320 ttttgatttg gaactctaat ttgaataagcagagccgttc aatgtcagtt tctactatat 1380 gattaaataa tcggtcctta attgtaatgcatcattcttt tttttttttt aactgaatcc 1440 ttgttccatg ctgtatgaac tcctttgagttccatttgta tatggtgctc ttgccattat 1500 aagagtagtg tttggtccaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaa 1553 6 368 PRT Oryza sativa 6 Ala Arg Ala SerPro Pro His Pro Lys Lys Phe Pro Thr Ile Pro Ser 1 5 10 15 Leu Ser LeuSer Asn Leu Asp Leu Ser Leu Ser Ser Pro Leu Val Phe 20 25 30 Ala Arg AlaSer Arg Val Val Val Val Gly Gly Gly Ala Met Ala Gly 35 40 45 Arg Ser GluLeu Val Val Ser Phe Gly Glu Met Leu Ile Asp Phe Val 50 55 60 Pro Thr ValAla Gly Val Ser Leu Ala Glu Ala Pro Ala Phe Val Lys 65 70 75 80 Ala ProGly Gly Ala Pro Ala Asn Val Ala Ile Ala Val Ala Arg Leu 85 90 95 Gly GlyGly Ala Ala Phe Val Gly Lys Leu Gly Asp Asp Glu Phe Gly 100 105 110 ArgMet Leu Ala Ala Ile Leu Arg Asp Asn Gly Val Asp Asp Gly Gly 115 120 125Val Val Phe Asp Ala Gly Ala Arg Thr Ala Leu Ala Phe Val Thr Leu 130 135140 Arg Ala Asp Gly Glu Arg Glu Phe Met Phe Tyr Arg Asn Pro Ser Ala 145150 155 160 Asp Met Leu Leu Thr His Ala Glu Leu Asn Val Glu Leu Ile LysArg 165 170 175 Ala Ala Val Phe His Tyr Gly Ser Ile Ser Leu Ile Ala GluPro Cys 180 185 190 Arg Ser Ala His Leu Arg Ala Met Glu Ile Ala Lys GluAla Gly Ala 195 200 205 Leu Leu Ser Tyr Asp Pro Asn Leu Arg Glu Ala LeuTrp Pro Ser Arg 210 215 220 Glu Glu Ala Arg Thr Lys Ile Leu Ser Ile TrpAsp Gln Ala Asp Ile 225 230 235 240 Val Lys Val Ser Glu Val Glu Leu GluPhe Leu Thr Gly Ile Asp Ser 245 250 255 Val Glu Asp Asp Val Val Met LysLeu Trp Arg Pro Thr Met Lys Leu 260 265 270 Leu Leu Val Thr Leu Gly AspGln Gly Cys Lys Tyr Tyr Ala Arg Asp 275 280 285 Phe Arg Gly Ala Val ProSer Tyr Lys Val Gln Gln Val Asp Thr Thr 290 295 300 Gly Ala Gly Asp AlaPhe Val Gly Ala Leu Leu Arg Arg Ile Val Gln 305 310 315 320 Asp Pro SerSer Leu Gln Asp Gln Lys Lys Leu Glu Glu Ala Ile Lys 325 330 335 Phe AlaAsn Ala Cys Gly Ala Ile Thr Ala Thr Lys Lys Gly Ala Ile 340 345 350 ProSer Leu Pro Thr Glu Val Glu Val Leu Lys Leu Met Glu Ser Ala 355 360 3657 1310 DNA Glycine max 7 gcacgagaga actagtctct cgtgccgctc gaaaacagtgttccaaaatc caaacacact 60 ctctctcccc atggcgttga acaatggcgt ccccgccaccggcaccggcc tcatcgtcag 120 cttcggtgag atgctcatcg acttcgtccc caccgtctctggcgtgtccc tggccgaggc 180 ccctggcttc ctcaaggccc ccggcggcgc ccccgctaacgtcgccatcg ccgtgtcgcg 240 cctcggcggc aaagccgcct tcgtcggcaa gctcggcgacgacgagttcg gccacatgct 300 cgccggaatc ctcaaggaaa acggcgttcg cgccgacggcatcaactttg accagggcgc 360 acgcaccgcc ctggccttcg tgaccctacg cgccgacggggagcgtgagt tcatgttcta 420 cagaaacccc agcgccgaca tgctcctcaa gcccgaagaactcaatctcg aactcatcag 480 atctgcaaaa gttttccatt acggatcaat cagtttgatcgtggagccat gcagatcagc 540 acacttgaag gcaatggaag tagccaagga atctgggtgcttgctctcct atgaccccaa 600 ccttcgtcta cctttgtggc cttcggctga ggaagctcgtaagcaaatac tgagcatttg 660 ggagaaggct gatttgatca aggtcagtga tgcggagcttgagttcctca caggaagtga 720 caagattgat gatgaatctg ctttgtcatt gtggcaccccaatttgaagt tgctccttgt 780 cactcttggg gaacatggtt ccagatacta caccaagagtttcaaaggat cggtagatgc 840 tttccatgtc aatacagttg atacaactgg tgccggtgattcctttgttg gtgctttatt 900 ggccaagatt gtcgatgatc agtccatact tgaagatgaaccaaggttaa gagaagtact 960 aaagtttgca aatgcatgtg gagctattac aactacccaaaagggagcaa ttccggccct 1020 tcccaaagag gaggctgcac tgaaactgat caaaggggggtcatagaatc ttttggcaaa 1080 atgcaaaagt gctagcatga tttcgttttc ttcccctaatgtttaaattt tccgttggat 1140 ttgcttgcta taagtttagg agggaacttt tgttttttctcctatgcact gttttcaggt 1200 tttgccaaat aacgctttct ttcaaatttt gagattagcgattgaatgaa aatttgaatc 1260 ataagctcgg cccatagttg caacttaaaa aaaaaaaaaaaaaaaaaaaa 1310 8 354 PRT Glycine max 8 His Glu Arg Thr Ser Leu Ser CysArg Ser Lys Thr Val Phe Gln Asn 1 5 10 15 Pro Asn Thr Leu Ser Leu ProMet Ala Leu Asn Asn Gly Val Pro Ala 20 25 30 Thr Gly Thr Gly Leu Ile ValSer Phe Gly Glu Met Leu Ile Asp Phe 35 40 45 Val Pro Thr Val Ser Gly ValSer Leu Ala Glu Ala Pro Gly Phe Leu 50 55 60 Lys Ala Pro Gly Gly Ala ProAla Asn Val Ala Ile Ala Val Ser Arg 65 70 75 80 Leu Gly Gly Lys Ala AlaPhe Val Gly Lys Leu Gly Asp Asp Glu Phe 85 90 95 Gly His Met Leu Ala GlyIle Leu Lys Glu Asn Gly Val Arg Ala Asp 100 105 110 Gly Ile Asn Phe AspGln Gly Ala Arg Thr Ala Leu Ala Phe Val Thr 115 120 125 Leu Arg Ala AspGly Glu Arg Glu Phe Met Phe Tyr Arg Asn Pro Ser 130 135 140 Ala Asp MetLeu Leu Lys Pro Glu Glu Leu Asn Leu Glu Leu Ile Arg 145 150 155 160 SerAla Lys Val Phe His Tyr Gly Ser Ile Ser Leu Ile Val Glu Pro 165 170 175Cys Arg Ser Ala His Leu Lys Ala Met Glu Val Ala Lys Glu Ser Gly 180 185190 Cys Leu Leu Ser Tyr Asp Pro Asn Leu Arg Leu Pro Leu Trp Pro Ser 195200 205 Ala Glu Glu Ala Arg Lys Gln Ile Leu Ser Ile Trp Glu Lys Ala Asp210 215 220 Leu Ile Lys Val Ser Asp Ala Glu Leu Glu Phe Leu Thr Gly SerAsp 225 230 235 240 Lys Ile Asp Asp Glu Ser Ala Leu Ser Leu Trp His ProAsn Leu Lys 245 250 255 Leu Leu Leu Val Thr Leu Gly Glu His Gly Ser ArgTyr Tyr Thr Lys 260 265 270 Ser Phe Lys Gly Ser Val Asp Ala Phe His ValAsn Thr Val Asp Thr 275 280 285 Thr Gly Ala Gly Asp Ser Phe Val Gly AlaLeu Leu Ala Lys Ile Val 290 295 300 Asp Asp Gln Ser Ile Leu Glu Asp GluPro Arg Leu Arg Glu Val Leu 305 310 315 320 Lys Phe Ala Asn Ala Cys GlyAla Ile Thr Thr Thr Gln Lys Gly Ala 325 330 335 Ile Pro Ala Leu Pro LysGlu Glu Ala Ala Leu Lys Leu Ile Lys Gly 340 345 350 Gly Ser 354 9 1736DNA Glycine max 9 ggaaaaaaga tgcagagcac acatgaaata tatagcatac actcctaagtttttttacaa 60 caaagtattg ttctagtata cgacacaaac cgcaaagtca aattctaaacaaaattagta 120 tacatcatag tactccaaaa actagaaatc actgaaagtt ctttcgattagcttccgaac 180 cactagtgga tggcacggcc actcctggcg ctacaaaatg tgagataccttctgagatgt 240 tcttaactgc tttgtcaaaa ttaacaaaac ccatgaacca aaaatcatgcccttccaccg 300 ttacaacctg aatgtacttt tctgatgggt tttccctcat ggtcactgggttgaccactc 360 caacctttcc caaaggcacc attaccttgt agtatgtcca agtttcttggccagagggtg 420 cagtgaaaca caaagggcga tcgctgcaaa acgctacatg aatattggacaggtaaaggg 480 ttcctgcaac aggacctgtt gatgttgaaa ggtaacaagc aaagctcttcttgagcttct 540 cgtttggata ggttgtgaat gtttgcttgt aaagggactc aaatccaccctctgatattg 600 ctttcacagt cagattcatc ttccccagtg cagctgaaga cactgatggaccagttttaa 660 ggttgtgcca gacgttgtgt gcggtggctt cagctttttt gctccatgaatcgaacatgt 720 taaggattga ctccatggga ctgttgcttg gtttgtcaac tgggctatgttgcacgtagg 780 gatgttgatg ttggtcatgg tagtattgaa ctggttgagg ttgtccactttgtaaagctg 840 cttttttgtt atctgggtgg ctgcttggaa cagcaggggt gcccattatgtgagttcccc 900 aattctcagt cccaacattg ggtggagaag atgatgatga tgatgctccttgtgcttctg 960 gaaatgattg gtttttttcg gtgtcgttgt tggtattcat cttggattgttagtgcaaga 1020 gaaaagaggt agaattagaa gcattcttct gcaattcaaa tcaaattttcaaaccatggc 1080 ttcctccacc aacgctcttc ctcccaccgg caacggcctc atcgtgagcttcggcgagat 1140 gctcatcgat ttcgtcccca ccgtctccgg cgtgtccctt gcggaggctccgggcttcct 1200 caaggccccc ggcggcgccc ccgccaacgt cgccatcgcc gtcgcgaggctcggcggaaa 1260 ggcggcgttc gtcggaaagc tcggcgacga cgagttcggg cacatgctggctggaatcct 1320 gaaggagaac gacgtgcgat ccgacgggat caacttcgac cagggcgcgcgcaccgcgct 1380 ggcgttcgtg accctacgcg ccgacggaga gcgtgagttc atgttctacagaaaccccag 1440 cgccgacatg ctcctcacgc ccgaagatct caatctcgaa ctcatcagatctgcaaaagt 1500 attccattat ggatcgataa gcttgatcgt ggagccatgc agatcagcacacctgaaggc 1560 aatggaagtt gccagggaag caggatgctt gctctcttat gacccaaacctgcggctacc 1620 cttgtggccc tccgccgagg aagcacgtca gcaaatactc agcatatgggacaaggctga 1680 tgtaatcaag gtcagtgatg tggaactgga attcctaacc ggaagtgacctcgtgc 1736 10 256 PRT Glycine max 10 Leu Val Phe Phe Gly Val Val ValGly Ile His Leu Gly Leu Leu Val 1 5 10 15 Gln Glu Lys Arg Gly Arg IleArg Ser Ile Leu Leu Gln Phe Lys Ser 20 25 30 Asn Phe Gln Thr Met Ala SerSer Thr Asn Ala Leu Pro Pro Thr Gly 35 40 45 Asn Gly Leu Ile Val Ser PheGly Glu Met Leu Ile Asp Phe Val Pro 50 55 60 Thr Val Ser Gly Val Ser LeuAla Glu Ala Pro Gly Phe Leu Lys Ala 65 70 75 80 Pro Gly Gly Ala Pro AlaAsn Val Ala Ile Ala Val Ala Arg Leu Gly 85 90 95 Gly Lys Ala Ala Phe ValGly Lys Leu Gly Asp Asp Glu Phe Gly His 100 105 110 Met Leu Ala Gly IleLeu Lys Glu Asn Asp Val Arg Ser Asp Gly Ile 115 120 125 Asn Phe Asp GlnGly Ala Arg Thr Ala Leu Ala Phe Val Thr Leu Arg 130 135 140 Ala Asp GlyGlu Arg Glu Phe Met Phe Tyr Arg Asn Pro Ser Ala Asp 145 150 155 160 MetLeu Leu Thr Pro Glu Asp Leu Asn Leu Glu Leu Ile Arg Ser Ala 165 170 175Lys Val Phe His Tyr Gly Ser Ile Ser Leu Ile Val Glu Pro Cys Arg 180 185190 Ser Ala His Leu Lys Ala Met Glu Val Ala Arg Glu Ala Gly Cys Leu 195200 205 Leu Ser Tyr Asp Pro Asn Leu Arg Leu Pro Leu Trp Pro Ser Ala Glu210 215 220 Glu Ala Arg Gln Gln Ile Leu Ser Ile Trp Asp Lys Ala Asp ValIle 225 230 235 240 Lys Val Ser Asp Val Glu Leu Glu Phe Leu Thr Gly SerAsp Leu Val 245 250 255 11 1348 DNA Triticum aestivum 11 gcacgaggcctcgtgccgaa tcgcacgagg ccgtcgcgtt cgcgtttccg tttcgtgcgt 60 ttagaccaagcttccaatgg ctcctctcgg tgacgctgtt gcccccgcgg cggccgccgc 120 cgcccctggcctcgtcgtct ctttcggcga gatgctgatc gacttcgtgc ctgacgttgc 180 cggcgtttccctcgccgagt ccggcggttt cgtcaaggcc cccggcggcg cccccgccaa 240 cgtcgcctgcgccatctcca agctcggcgg ctcctccgcc ttcatcggaa agtttggcga 300 cgacgagttcggccacatgc tggtggagat cctgaagcag aacggcgtaa acgccgaggg 360 ctgcctgttcgaccagcacg cgcgcaccgc gctggccttc gtcacgctca agtccaacgg 420 cgagcgcgagttcatgttct accgcaaccc gtcggccgac atgttgctca ccgaggccga 480 gctcaacctggacctgatcc gccgcgcccg catcttccac tacggctcca tctcgctcat 540 caccgagccctgccgctcgg cccacgtcgc cgccacgcgt gccgccaagt cggccggcat 600 cctttgttcgtacgacccca acgtgcgcct gccgctctgg ccctctgcgc aggccgcccg 660 cgacggcatcatgagcatct ggaaggaggc tgacttcatc aaggtgagcg acgaggaggt 720 agccttcctcacccagggcg acgccactga cgagaagaac gtgctctccc tctggttcga 780 gggcctcaagctgctcatcg tcaccgatgg tgagaagggg tgcaggtact tcaccaagga 840 cttcaagggctcggtgcccg gctactctgt caacaccgtg gacaccaccg gcgccggcga 900 cgccttcgtcggctccctcc tcgtcagcgt ctccaaggac gactccatct tctacaatga 960 ggccaagctgagggaggtgc tgcagttctc gaacgcttgc ggcgccatct gcaccaccaa 1020 gaagggagccatcccggcgc tgcccaccac cgccaccgcc ctggagctca tcagcaaggg 1080 cagtaactagagactcattg tgtcgcgcca tcggggttga atcttaggag ttttagctgc 1140 acttttattattattattag ggatgaattg agtttagttc gtgagtcaag tgtgtgtgac 1200 ctcgtgggcgcttaataaaa agcaagcatg tgtggtgatt ttggttgcgg tctttgtgta 1260 aggaggctactgattgttgt agccttcacc caaactttat cagagtcttt aatgaatgga 1320 caagttcatcaaaaaaaaaa aaaaaaaa 1348 12 337 PRT Triticum aestivum 12 Met Ala Pro LeuGly Asp Ala Val Ala Pro Ala Ala Ala Ala Ala Ala 1 5 10 15 Pro Gly LeuVal Val Ser Phe Gly Glu Met Leu Ile Asp Phe Val Pro 20 25 30 Asp Val AlaGly Val Ser Leu Ala Glu Ser Gly Gly Phe Val Lys Ala 35 40 45 Pro Gly GlyAla Pro Ala Asn Val Ala Cys Ala Ile Ser Lys Leu Gly 50 55 60 Gly Ser SerAla Phe Ile Gly Lys Phe Gly Asp Asp Glu Phe Gly His 65 70 75 80 Met LeuVal Glu Ile Leu Lys Gln Asn Gly Val Asn Ala Glu Gly Cys 85 90 95 Leu PheAsp Gln His Ala Arg Thr Ala Leu Ala Phe Val Thr Leu Lys 100 105 110 SerAsn Gly Glu Arg Glu Phe Met Phe Tyr Arg Asn Pro Ser Ala Asp 115 120 125Met Leu Leu Thr Glu Ala Glu Leu Asn Leu Asp Leu Ile Arg Arg Ala 130 135140 Arg Ile Phe His Tyr Gly Ser Ile Ser Leu Ile Thr Glu Pro Cys Arg 145150 155 160 Ser Ala His Val Ala Ala Thr Arg Ala Ala Lys Ser Ala Gly IleLeu 165 170 175 Cys Ser Tyr Asp Pro Asn Val Arg Leu Pro Leu Trp Pro SerAla Gln 180 185 190 Ala Ala Arg Asp Gly Ile Met Ser Ile Trp Lys Glu AlaAsp Phe Ile 195 200 205 Lys Val Ser Asp Glu Glu Val Ala Phe Leu Thr GlnGly Asp Ala Thr 210 215 220 Asp Glu Lys Asn Val Leu Ser Leu Trp Phe GluGly Leu Lys Leu Leu 225 230 235 240 Ile Val Thr Asp Gly Glu Lys Gly CysArg Tyr Phe Thr Lys Asp Phe 245 250 255 Lys Gly Ser Val Pro Gly Tyr SerVal Asn Thr Val Asp Thr Thr Gly 260 265 270 Ala Gly Asp Ala Phe Val GlySer Leu Leu Val Ser Val Ser Lys Asp 275 280 285 Asp Ser Ile Phe Tyr AsnGlu Ala Lys Leu Arg Glu Val Leu Gln Phe 290 295 300 Ser Asn Ala Cys GlyAla Ile Cys Thr Thr Lys Lys Gly Ala Ile Pro 305 310 315 320 Ala Leu ProThr Thr Ala Thr Ala Leu Glu Leu Ile Ser Lys Gly Ser 325 330 335 Asn 33713 328 PRT Lycopersicon esculentum 13 Met Ala Val Asn Gly Ala Ser SerSer Gly Leu Ile Val Ser Phe Gly 1 5 10 15 Glu Met Leu Ile Asp Phe ValPro Thr Val Ser Gly Val Ser Leu Ala 20 25 30 Glu Ala Pro Gly Phe Leu LysAla Pro Gly Gly Ala Pro Ala Asn Val 35 40 45 Ala Ile Ala Val Thr Arg LeuGly Gly Lys Ser Ala Phe Val Gly Lys 50 55 60 Leu Gly Asp Asp Glu Phe GlyHis Met Leu Ala Gly Ile Leu Lys Thr 65 70 75 80 Asn Gly Val Gln Ala GluGly Ile Asn Phe Asp Lys Gly Ala Arg Thr 85 90 95 Ala Leu Ala Phe Val ThrLeu Arg Ala Asp Gly Glu Arg Glu Phe Met 100 105 110 Phe Tyr Arg Asn ProSer Ala Asp Met Leu Leu Thr Pro Ala Glu Leu 115 120 125 Asn Leu Asp LeuIle Arg Ser Ala Lys Val Phe His Tyr Gly Ser Ile 130 135 140 Ser Leu IleVal Glu Pro Cys Arg Ala Ala His Met Lys Ala Met Glu 145 150 155 160 ValAla Lys Glu Ala Gly Ala Leu Leu Ser Tyr Asp Pro Asn Leu Arg 165 170 175Leu Pro Leu Trp Pro Ser Ala Glu Glu Ala Lys Lys Gln Ile Lys Ser 180 185190 Ile Trp Asp Ser Ala Asp Val Ile Lys Val Ser Asp Val Glu Leu Glu 195200 205 Phe Leu Thr Gly Ser Asn Lys Ile Asp Asp Glu Ser Ala Met Ser Leu210 215 220 Trp His Pro Asn Leu Lys Leu Leu Leu Val Thr Leu Gly Glu LysGly 225 230 235 240 Cys Asn Tyr Tyr Thr Lys Lys Phe His Gly Thr Val GlyGly Phe His 245 250 255 Val Lys Thr Val Asp Thr Thr Gly Ala Gly Asp SerPhe Val Gly Ala 260 265 270 Leu Leu Thr Lys Ile Val Asp Asp Gln Thr IleLeu Glu Asp Glu Ala 275 280 285 Arg Leu Lys Glu Val Leu Arg Phe Ser CysAla Cys Gly Ala Ile Thr 290 295 300 Thr Thr Lys Lys Gly Ala Ile Pro AlaLeu Pro Thr Ala Ser Glu Ala 305 310 315 320 Leu Thr Leu Leu Lys Gly GlyAla 325

What is claimed is:
 1. An isolated polynucleotide comprising: (a) afirst nucleotide sequence encoding a first polypeptide havingfructokinase activity, wherein the amino acid sequence of the firstpolypeptide and the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6,or SEQ ID NO: 12 have at least 80% identity based on the Clustalalignment method, (b) a second nucleotide sequence encoding a secondpolypeptide having fructokinase activity, wherein the amino acidsequence of the second polypeptide and the amino acid sequence of SEQ IDNO: 8 or SEQ ID NO: 10 have at least 90% identity based on the Clustalalignment method, (c) a third nucleotide sequence encoding a thirdpolypeptide having fructokinase activity, wherein the amino acidsequence of the third polypeptide and the amino acid sequence of SEQ IDNO: 4 have at least 95% identity based on the Clustal alignment method,or (d) the complement of the first, second, or third nucleotidesequence, wherein the complement and the first, second, or thirdnucleotide sequence contain the same number of nucleotides and are 100%complementary.
 2. The polynucleotide of claim 1, wherein the amino acidsequence of the first polypeptide and the amino acid sequence of SEQ IDNO: 2, SEQ ID NO: 6, or SEQ ID NO: 12 have at least 85% identity basedon the Clustal alignment method.
 3. The polynucleotide of claim 1,wherein the amino acid sequence of the first polypeptide and the aminoacid sequence of SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 12 have atleast 90% identity based on the Clustal alignment method.
 4. Thepolynucleotide of claim 1, wherein the amino acid sequence of the firstpolypeptide and the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6,or SEQ ID NO: 12 have at least 95% identity based on the Clustalalignment method, and wherein the amino acid sequence of the secondpolypeptide and the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10have at least 95% identity based on the Clustal alignment method.
 5. Thepolynucleotide of claim 1, wherein the first polypeptide comprises theamino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 12,wherein the second polypeptide comprises the amino acid sequence of SEQID NO: 8 or SEQ ID NO: 10, and wherein the third polypeptide comprisesthe amino acid sequence of SEQ ID NO:
 4. 6. The polynucleotide of claim1, wherein the first nucleotide sequence comprises the nucleotidesequence of SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 11, wherein thesecond nucleotide sequence comprises the nucleotide sequence of SEQ IDNO: 7 or SEQ ID NO: 9, and wherein the third nucleotide sequencecomprises the nucleotide sequence of SEQ ID NO:
 3. 7. An vectorcomprising the polynucleotide of claim
 1. 8. A recombinant DNA constructcomprising the polynucleotide of claim 1 operably linked to a regulatorysequence.
 9. A method for transforming a cell comprising transforming acell with the polynucleotide of claim
 1. 10. A cell comprising therecombinant DNA construct of claim
 8. 11. A method for producing a plantcomprising transforming a plant cell with the polynucleotide of claim 1and regenerating a plant from the transformed plant cell.
 12. A plantcomprising the recombinant DNA construct of claim
 8. 13. A seedcomprising the recombinant DNA construct of claim
 8. 14. An isolatedpolynucleotide comprising a first nucleotide sequence, wherein the firstnucleotide sequence contains at least 30 nucleotides, and wherein thefirst nucleotide sequence is comprised by another polynucleotide,wherein the other polynucleotide includes: (a) a second nucleotidesequence, wherein the second nucleotide sequence encodes a polypeptidehaving fructokinase activity, wherein the amino acid sequence of thepolypeptide and the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6,or SEQ ID NO: 12 have at least 80% sequence identity based on theClustal alignment method, or (b) the complement of the second nucleotidesequence, wherein the complement and the second nucleotide sequencecontain the same number of nucleotides and are 100% complementary. 15.An isolated polynucleotide comprising a first nucleotide sequence,wherein the first nucleotide sequence contains at least 30 nucleotides,and wherein the first nucleotide sequence is comprised by anotherpolynucleotide, wherein the other polynucleotide includes: (a) a secondnucleotide sequence, wherein the second nucleotide sequence encodes apolypeptide having fructokinase activity, wherein the amino acidsequence of the polypeptide and the amino acid sequence of SEQ ID NO: 8or SEQ ID NO: 10 have at least 90% sequence identity based on theClustal alignment method, or (b) the complement of the second nucleotidesequence, wherein the complement and the second nucleotide sequencecontain the same number of nucleotides and are 100% complementary. 16.An isolated polynucleotide comprising a first nucleotide sequence,wherein the first nucleotide sequence contains at least 30 nucleotides,and wherein the first nucleotide sequence is comprised by anotherpolynucleotide, wherein the other polynucleotide includes: (a) a secondnucleotide sequence, wherein the second nucleotide sequence encodes apolypeptide having fructokinase activity, wherein the amino acidsequence of the polypeptide and the amino acid sequence of SEQ ID NO: 4have at least 95% sequence identity based on the Clustal alignmentmethod, or (b) the complement of the second nucleotide sequence, whereinthe complement and the second nucleotide sequence contain the samenumber of nucleotides and are 100% complementary.
 17. An isolatedpolypeptide having fructokinase activity wherein the polypeptidecomprises: (a) a first amino acid sequence, wherein the first amino acidsequence and the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, orSEQ ID NO: 12 have at least 80% identity based on the Clustal alignment,(b) a second amino acid sequence, wherein the second amino acid sequenceand the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10 have atleast 90% identity based on the Clustal alignment method, or (c) a thirdamino acid sequence, wherein the third amino acid sequence and the aminoacid sequence of SEQ ID NO: 4 have at least 95% identity based on theClustal alignment method.
 18. The polypeptide of claim 17, wherein thefirst amino acid sequence and the amino acid sequence of SEQ ID NO: 2,SEQ ID NO: 6, or SEQ ID NO: 12 have at least 85% identity based on theClustal alignment method.
 19. The polypeptide of claim 17, wherein thefirst amino acid sequence and the amino acid sequence of SEQ ID NO: 2,SEQ ID NO: 6, or SEQ ID NO: 12 have at least 90% identity based on theClustal alignment method.
 20. The polypeptide of claim 17, wherein thefirst amino acid sequence and the amino acid sequence of SEQ ID NO: 2,SEQ ID NO: 6, or SEQ ID NO: 12 have at least 95% identity based on theClustal alignment method, and wherein the second amino acid sequence andthe amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10 have at least95% identity based on the Clustal alignment method.
 21. The polypeptideof claim 17, wherein the first amino acid comprises the amino acidsequence of SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 12, wherein thesecond amino acid sequence comprises the amino acid sequence of SEQ IDNO:8 or SEQ ID NO: 10, and wherein the third amino acid comprises theamino acid sequence of SEQ ID NO:
 4. 22. A method for isolating apolypeptide encoded by the polynucleotide of claim 1 comprisingisolating the polypeptide from a cell containing a recombinant DNAconstruct comprising the polynucleotide operably linked to a regulatorysequence.